The selection of mutants using cultured plant cells is in principle similar to that done with microorganisms, but in practice is much more difficult. The reasons for the difficulties include the usual clumpy nature of plant cell cultures; single cells or protoplasts usually cannot be easily grown to form clones, cell growth is slow and the cells are usually not monoploid. Despite these problems a large number of successful selection experiments have been carried out to produce mutants of value for producing compounds, for biochemical and molecular biology studies, for markers in genetic experiments and for improving crop plants. Part of the reason for the success is that cell systems allow the screening of millions of cells for the desired trait.
Whether the selected phenotype is under genetic or epigenetic control can most easily be determined by regenerating plants and by following the phenotype in progeny. Genetically controlled phenotypes would be inherited by progeny and would generally be more stable at the cell level in comparison to epigenetically controlled traits. A large number of in vitro selected traits have been shown to be expressed in regenerated plants and to be passed on to progeny.
There are several types of in vitro selection that can be used to obtain cells containing the trait of interest (J. Widholm, Iowa State J. of Research, 62: 587-597, 1988). These include selection for growth, selection for valuable compound production, auxotroph selection and resistance selection. Selection for resistance should be the easiest kind of selection to accomplish and from the number of reports in the literature this would appear to be true.
The selection for amino acid analog resistance in plants has been pursued for a number of years. A primary focus of this research has been directed to the enzyme anthranilate synthase (AS). AS catalyzes the conversion of chorismate into anthranilate, the first reaction leading from the common aromatic amino acid (shikimate) pathway toward the biosynthesis of tryptophan (Trp). As a branchpoint enzyme in the synthesis of aromatic amino acids, AS plays a key role in the diversion of chorismate into Trp and indolic secondary compound biosynthesis.
Available information indicates that AS plays a key role in regulation of Trp biosynthesis. In plants, bacteria, and fungi, AS activity is regulated by Trp feedback inhibition (Matsui et al., J. Bacteriol, 169: 5330-5332, 1987). In microbes, AS usually consists of two nonidentical subunits, referred to as the alpha subunit (component I) and the beta subunit (component II). Component I can convert chorismate to anthranilate in the presence of high levels of ammonia (ammonia-dependent AS activity), whereas component II is responsible for the use of Gln as the amino donor (Hutter et al., Annu Rev Microbiol, 40: 55-77, 1986).
As a means to investigate regulation of the Trp pathway, toxic analogs of Trp have been used in metabolic studies of plant cell cultures and as a tool to select mutants. Many of these studies have been conducted with the growth inhibitor 5-methyltryptophan (5MT). In a number of species including Datura innoxia (hereinafter referred to as D. innoxia), Catharanthus roseus, and Solanum tuberosum, variant cell lines resistant to inhibitory concentrations of 5MT were found to have AS that was less sensitive to feedback inhibition by Trp (Carlson and Widholm, Physiol Plant, 44: 251-255, 1978; Scott et al., Phytochemistry, 18: 795-798, 1979; Ranch et al., Plant Physiol, 71: 136-140, 1983). Widholm (Planta, 134: 103-108, 1977) described 5-methyltryptophan-resistant carrot cell lines and a potato cell line that were auxin autotrophic.
In addition, 5-methylanthranilate was successfully used to isolate plant auxotrophic mutants defective in three different genes, trp1, trp2, and trp3 (Last and Fink, Science, 240: 305-310, 1988; Last et al., Plant Cell, 3: 345-358, 1991) and mutants of Chlamydomonas reinhardtii (Dutcher et al., Genetics, 131: 593-607, 1992). Mutants resistant to 5MT or alpha-methyltryptophan (.alpha.MT) were reported in Arabidopsis thaliana (hereinafter referred to as A. thaliana) (Koornneef and van Loenen Martinet, Arabidopsis Inf Serv, 20: 104-108, 1983; Kreps & Town, Plant Physiol, 99: 269-275, 1992), maize (Kang & Kameya, Euphytica, 69: 95-101, 1993), Lemna gibba (Tam et al., Plant Physiol, 107: 77-85, 1995) and Oryza sativa (Lee & Kameya, Theor Appl Genet, 82: 405-408, 1991). The specificity of selection with these analogs have not been systematically investigated.
A feedback-insensitive AS gene (ASA1 mutant) has been recently obtained by selection of mutagenized Arabidopsis seeds resistant to 6-methylanthranilate (Li & Last, Plant Physiol., 110: 51-59, 1996). In addition, .alpha.MT resistance led to identification of a mutant in A. thaliana with the same amino acid change (Kreps et al., Plant Physiol., 110: 1159-1165, 1996).
One method for the production of transgenic plants is to transform plant cells in tissue culture with a plasmid containing a promoter and selectable marker which also contains a gene which would express the desired trait in the regenerated plant. Thus when one selects cells transformed with the selectable marker, many of these cells will also carry the gene that will also be expressed to produce the desired result such as insect resistance, disease resistance, herbicide resistance, changed starch, drought tolerance, etc. An example is where the nptII (neo) gene is driven by a constitutive promoter, nosP (Vermeulen et al., Plant Cell Reports, 11: 243-247, 1992). Next to this selectable marker gene is a mutant acetolactate synthase gene with its own promoter. This latter gene makes the regenerated plants resistant to certain herbicides.
The AS gene which encodes for an enzyme that is highly resistant to an amino acid analog, such as 5MT, would be an ideal selectable marker for the production of transgenic plants as described above. Especially if the promoter which regulates the expression of this enzyme provided for high level expression of the enzyme in tissue culture, and little or no expression in regenerated plants. There has been considerable environmental concern because most selectable markers are constitutively expressed in all tissues of the plant and are not of plant origin. The former concern would be reduced by using such a tissue culture specific promoter while the latter concern would be eliminated by using the plant-derived AS gene as the selectable marker. In fact, the use of a tissue culture specific promoter would even allow one to use selectable markers that are not of plant origin. Traditional selectable markers that are not of plant origin include nptII, which encodes kanamycin resistance.